Adrenomedullin assays and methods for determining mature adrenomedullin

ABSTRACT

Subject of the present invention is an in vitro method for therapy follow-up in septic patients wherein the concentration of mature ADM 1-52 and/or mature ADM 1-52-Gly in a sample of bodily fluid of said septic patient is determined using an assay comprising two binders that bind to two different regions within the region of mature adrenomedullin and/or adrenomedullin-Gly that is aminoacid 21-52-amid SEQ ID No. 1 or aminoacid 21-52-Gly SEQ ID No. 2 wherein each of said regions comprises at least 4 or 5 amino acids. 
     Subject of the present invention are further assays and calibration methods.

Subject of the present invention is an in vitro method for therapyfollow-up in septic patients wherein the concentration of mature ADM1-52 and/or mature ADM 1-52-Gly in a sample of bodily fluid of saidseptic patient is determined using an assay comprising two binders thatbind to two different regions within the region of mature adrenomedullinand/or adrenomedullin-Gly that is aminoacid 21-52-amid SEQ ID No. 1 oraminoacid 21-52-Gly SEQ ID No. 2 wherein each of said regions comprisesat least 4 or 5 amino acids.

Subject of the present invention are further assays and calibrationmethods.

The peptide adrenomedullin (ADM) was described for the first time inKitamura et al., (cf. 1; numerical data are based on the attached listof references) as a novel hypotensive peptide comprising 52 amino acids,which had been isolated from a human pheochromocytoma. In the same year,cDNA coding for a precursor peptide comprising 185 amino acids and thecomplete amino acid sequence of this precursor peptide were alsodescribed. The precursor peptide, which comprises, inter alia, a signalsequence of 21 amino acids at the N-terminus, is referred to as“preproadrenomedullin” (pre-proADM). Pre-proADM comprises 185 aminoacids and has the sequence according to SEQ ID No: 3. The mature ADM isdisplayed in SEQ ID No. 4 and the mature ADM-Gly is displayed in SEQ No.5.

The peptide adrenomedullin (ADM) is a peptide which comprises 52 aminoacids (SEQ ID No: 2) and which comprises the amino acids 95 to 146 ofpre-proADM, from which it is formed by proteolytic cleavage. To date,substantially only a few fragments of the peptide fragments formed inthe cleavage of the pre-proADM have been more exactly characterized, inparticular the physiologically active peptides adrenomedullin (ADM) and“PAMP”, a peptide comprising 20 amino acids (22-41) which follows the 21amino acids of the signal peptide in pre-proADM. For both ADM and PAMP,physiologically active sub-fragments have furthermore been discoveredand investigated in more detail. The discovery and characterization ofADM in 1993 triggered intensive research activity and a flood ofpublications, the results of which have recently been summarized invarious review articles, in the context of the present description,reference being made in particular to the articles to be found in anissue of “Peptides” devoted to ADM (Peptides 22 (2001)), in particular(2) and (3). A further review is (4). In the scientific investigationsto date, it has been found, inter alia, that ADM may be regarded as apolyfunctional regulatory peptide. It is released into the circulationin an inactive form extended by glycine (5). There is also a bindingprotein (6) which is specific for ADM and probably likewise modulatesthe effect of ADM.

Those physiological effects of ADM as well as of PAMP which are ofprimary importance in the investigations to date were the effectsinfluencing blood pressure. Thus, ADM is an effective vasodilator, itbeing possible to associate the hypotensive effect with in particularpeptide segments in the C-terminal part of ADM.

It has furthermore been found that the abovementioned furtherphysiologically active peptide PAMP formed from pre-proADM likewiseexhibits a hypotensive effect, even if it appears to have an actionmechanism differing from that of ADM (cf. in addition to theabovementioned review articles (3) and (4) also (7), (8) or (9) and(10)).

It has furthermore been found that the concentrations of ADM which canbe measured in the circulation and other biological fluids are, in anumber of pathological states, significantly above the concentrations tobe found in healthy control persons. Thus, the ADM level in patientswith congestive heart failure, myocardial infarction, kidney diseases,hypertensive disorders, Diabetes mellitus, in the acute phase of shockand in sepsis and septic shock are significantly increased, although todifferent extents. The PAMP concentrations are also increased in some ofsaid pathological states, but the plasma levels are reduced relative toADM ((3); page 1702).

It is furthermore known that unusually high concentrations of ADM are tobe observed in sepsis or in septic shock (cf. (3) and (11), (12), (13),(14) and (15)). The findings are related to the typical hemodynamicchanges which are known as typical phenomena of the course of a diseasein patients with sepsis and other severe syndromes, such as, forexample, SIRS.

Although it is assumed that ADM and PAMP are formed from the sameprecursor peptide, pre-proADM (SEQ ID) No: 3), in which the amino acidsequences corresponding to these peptides are present as partialpeptides in equimolar amounts, the concentrations of ADM or PAMPmeasurable in biological fluids apparently differ. This is nothingunusual.

Thus, the measurable concentrations of different degradation products ofone and the same precursor peptide may be different, for example,because they are the result of different competing degradation pathwayswhich, for example in the case of different pathological states, lead todifferent fragmentation of a precursor peptide and hence to differentdegradation products. Certain partial peptides contained in theprecursor peptide may be formed as free peptides or may not be formed,and/or different peptides are formed in different ways and in differentamounts. Even if only a single degradation pathway is taken forprocessing a precursor peptide, and hence all degradation productsoriginate from one and the same precursor peptide and must have beenformed per se primarily in equimolar amounts, the steady stateconcentrations of different partial peptides and fragments measurable inbiological fluids may be very different, namely, for example, whenindividual ones thereof are formed at a different rate and/or havedifferent individual stabilities (lifetimes) in the respectivebiological fluid, or if they are removed from circulation on the basisof different clearance mechanisms and/or at different clearance rates.

Adrenomedullin plays pivotal roles during sepsis development ((16),(17)) and in numerous acute and chronic diseases ((18), (4)).

ADM is elevated in Sepsis and prognostic for outcome in sepsis ((19),(14), (11)). Sepsis treatment follow up for early monitoring oftreatment success or failure is a substantial remaining unmet clinicalneed.

At present there are no ADM assays suitable for routine diagnostics. Thesensitivity of currently available tests to determine mature ADM is toolow. Therefore, high plasma volume is needed for analysis. Further,presently available assays exhibit stability related pre-analyticallimitations, for instance the samples need to be stabilized byAprotinin. ((20), (21)). On top, some ADM assays need an extensivesample preparation before measurement (11).

Goal of the present invention is to provide an assay that is suitable asa routine method for direct measurement of mature ADM suitable forstandard automated laboratory and point of care technologies.

Surprisingly, it turned out that such an assay may be used for treatmentfollow up in septic patients.

Subject of the present invention is an in vitro method for therapyfollow-up in septic patients wherein the concentration of mature ADM1-52 and/or mature ADM 1-52-Gly in a sample of bodily fluid of saidseptic patient is determined using an assay comprising two binders thatbind to two different regions within the region of mature adrenomedullinand/or adrenomedullin-Gly that is aminoacid 21-52-amid SEQ ID No. 1 oraminoacid 21-52-Gly SEQ ID No. 2 wherein each of said regions comprisesat least 4 or 5 amino acids.

In one embodiment of the invention subject is an in vitro method fortherapy follow-up in septic patients wherein one of said binders bindsto a region comprised within the following sequence of mature ADM and/ormature ADM 1-52-Gly:

-   -   ADM 21-32: CTVQKLAHQIYQ (SEQ ID No. 6)    -   and wherein said second of these binders binds to a region        comprised within the following sequence of mature ADM and/or        mature ADM 1-52-Gly:    -   ADM 42-52: APRSKISPQGY (SEQ ID No. 7)

In one embodiment of the invention the assay sensitivity of said assayis able to quantify the ADM of healthy subjects and is <10 pg/ml,preferably <40 pg/ml and more preferably <70 pg/ml.

In one embodiment of the invention said binder exhibits an bindingaffinity to mature ADM and/or mature ADM 1-52-Gly of at least 10⁷ M⁻¹,preferred 10⁸ M⁻¹, preferred affinity is greater than 10⁹ M⁻¹, mostpreferred greater than 10¹⁰ M⁻¹ A person skilled in the art knows thatit may be considered to compensate lower affinity by applying a higherdose of compounds and this measure would not lead out-of-the-scope ofthe invention.

To determine the affinity of the antibodies to Adrenomedullin, thekinetics of binding of Adrenomedullin to immobilized antibody wasdetermined by means of label-free surface plasmon resonance using aBiacore 2000 system (GE Healthcare Europe GmbH, Freiburg, Germany).Reversible immobilization of the antibodies was performed using ananti-mouse Fc antibody covalently coupled in high density to a CM5sensor surface according to the manufacturer's instructions (mouseantibody capture kit; GE Healthcare), (22).

In one embodiment of the invention said binder is selected from thegroup comprising an anti-adrenomedullin antibody or an anti-ADM antibodyfragment binding to ADM or a non-Ig scaffold binding to adrenomedullin.

Therapy follow up means at least once the concentration of ADM mature1-52 (SEQ ID No. 4) and/or mature ADM 1-52-Gly (SEQ ID No. 5) isdetermined in a sample, preferably more than once, preferably twice oronce a day after the start of therapy.

In one embodiment of the invention it may be a so-called POC-test(point-of-care), that is a test technology which allows performing thetest within less than 1 hour near the patient without the requirement ofa fully automated assay system. One example for this technology is theimmunochromatographic test technology.

In one embodiment of the invention such an assay is a sandwichimmunoassay using any kind of detection technology including but notrestricted to enzyme label, chemiluminescence label,electrochemiluminescence label, preferably a fully automated assay. Inone embodiment of the invention such an assay is an enzyme labeledsandwich assay. Examples of automated or fully automated assay compriseassays that may be used for one of the following systems: RocheElecsys®, Abbott Architect®, Siemens Centauer®, Brahms Kryptor®,Biomerieux Vidas®, Alere Triage®.

A variety of immunoassays are known and may be used for the assays andmethods of the present invention, these include: radioimmunoassays(“RIA”), homogeneous enzyme-multiplied immunoassays (“EMIT”), enzymelinked immunoadsorbent assays (“ELISA”), apoenzyme reactivationimmunoassay (“ARIS”), dipstick immunoassays and immuno-chromotographyassays.

In one embodiment of the invention at least one of said two binders islabeled in order to be detected.

The preferred detection methods comprise immunoassays in various formatssuch as for instance radioimmunoassay (RIA), chemiluminescence- andfluorescence-immunoassays, Enzyme-linked immunoassays (ELISA),Luminex-based bead arrays, protein microarray assays, and rapid testformats such as for instance immunochromatographic strip tests.

In a preferred embodiment said label is selected from the groupcomprising chemiluminescent label, enzyme label, fluorescence label,radioiodine label.

The assays can be homogenous or heterogeneous assays, competitive andnon-competitive assays. In one embodiment, the assay is in the form of asandwich assay, which is a non-competitive immunoassay, wherein themolecule to be detected and/or quantified is bound to a first antibodyand to a second antibody. The first antibody may be bound to a solidphase, e.g. a bead, a surface of a well or other container, a chip or astrip, and the second antibody is an antibody which is labeled, e.g.with a dye, with a radioisotope, or a reactive or catalytically activemoiety. The amount of labeled antibody bound to the analyte is thenmeasured by an appropriate method. The general composition andprocedures involved with “sandwich assays” are well-established andknown to the skilled person (23).

In another embodiment the assay comprises two capture molecules,preferably antibodies which are both present as dispersions in a liquidreaction mixture, wherein a first labelling component is attached to thefirst capture molecule, wherein said first labelling component is partof a labelling system based on fluorescence- orchemiluminescence-quenching or amplification, and a second labellingcomponent of said marking system is attached to the second capturemolecule, so that upon binding of both capture molecules to the analytea measurable signal is generated that allows for the detection of theformed sandwich complexes in the solution comprising the sample.

In another embodiment, said labeling system comprises rare earthcryptates or rare earth chelates in combination with fluorescence dye orchemiluminescence dye, in particular a dye of the cyanine type.

In the context of the present invention, fluorescence based assayscomprise the use of dyes, which may for instance be selected from thegroup comprising FAM (5- or 6-carboxyfluorescein), VIC, NED,Fluorescein, Fluoresceinisothiocyanate (FITC), IRD-700/800, Cyaninedyes, auch as CY3, CY5, CY3.5, CY5.5, Cy7, Xanthen,6-Carboxy-2′,4′,7′,4,7-hexachlorofluorescein (HEX), TET,6-Carboxy-4′,5′-dichloro-2′,7′-dimethodyfluorescein (JOE),N,N,N′,N′-Tetramethyl-6-carboxyrhodamine (TAMRA), 6-Carboxy-X-rhodamine(ROX), 5-Carboxyrhodamine-6G (R6G5), 6-carboxyrhodamine-6G (RG6),Rhodamine, Rhodamine Green, Rhodamine Red, Rhodamine 110, BODIPY dyes,such as BODIPY TMR, Oregon Green, Coumarines such as Umbelliferone,Benzimides, such as Hoechst 33258; Phenanthridines, such as Texas Red,Yakima Yellow, Alexa Fluor, PET, Ethidiumbromide, Acridinium dyes,Carbazol dyes, Phenoxazine dyes, Porphyrine dyes, Polymethin dyes, andthe like.

In the context of the present invention, chemiluminescence based assayscomprise the use of dyes, based on the physical principles described forchemiluminescent materials in (24). Preferred chemiluminescent dyes areacridiniumesters.

As mentioned herein, an “assay” or “diagnostic assay” can be of any typeapplied in the field of diagnostics. Such an assay may be based on thebinding of an analyte to be detected to one or more capture probes witha certain affinity. Concerning the interaction between capture moleculesand target molecules or molecules of interest, the affinity constant ispreferably greater than 10⁸ M⁻¹.

In the context of the present invention, “binder molecules” aremolecules which may be used to bind target molecules or molecules ofinterest, i.e. analytes (i.e. in the context of the present inventionPCT and fragments thereof), from a sample. Binder molecules must thus beshaped adequately, both spatially and in terms of surface features, suchas surface charge, hydrophobicity, hydrophilicity, presence or absenceof lewis donors and/or acceptors, to specifically bind the targetmolecules or molecules of interest. Hereby, the binding may for instancebe mediated by ionic, van-der-Waals, pi-pi, sigma-pi, hydrophobic orhydrogen bond interactions or a combination of two or more of theaforementioned interactions between the capture molecules and the targetmolecules or molecules of interest. In the context of the presentinvention, binder molecules may for instance be selected from the groupcomprising a nucleic acid molecule, a carbohydrate molecule, a PNAmolecule, a protein, an antibody, a peptide or a glycoprotein.Preferably, the binder molecules are antibodies, including fragmentsthereof with sufficient affinity to a target or molecule of interest,and including recombinant antibodies or recombinant antibody fragments,as well as chemically and/or biochemically modified derivatives of saidantibodies or fragments derived from the variant chain with a length ofat least 12 amino acids thereof.

Chemiluminescent label may be acridinium ester label, steroid labelsinvolving isoluminol labels and the like.

Enzyme labels may be lactate dehydrogenase (LDH), creatinekinase (CPK),alkaline phosphatase, aspartate aminotransferace (AST), alanineaminotransferace (ALT), acid phosphatase, glucose-6-phosphatedehydrogenase and so on.

In one embodiment of the invention at least one of said two binders isbound to a solid phase as magnetic particles, and polystyrene surfaces.

In one embodiment of the invention the concentration of mature ADM 1-52and/or mature ADM 1-52-Gly measured in the sample is in the rangebetween 10-500 pg/ml in plasma or blood.

The ADM levels of the present invention have been determined with thedescribed ADM assay. The above mentioned values might be different inother ADM assays, depending upon their way of calibration. The abovementioned values shall apply for such differently calibrated ADM assaysaccordingly, taking into account the differences in calibration. ADMassays could be calibrated by correlation and adjustment via theirnormal ranges (healthy population). Alternatively, commerciallyavailable control samples could be used for adjustment of differentcalibrations (ICI Diagnostics, Berlin, Germany). With the described ADMassay, the median of of a normal population has been determined to be24.7 pg/mL.

In one embodiment of the invention a threshold is applied whereby avalue above threshold is indicative of a patient that is not or badresponding to therapy and whereas a value below said threshold isindicative of a patient responding to therapy.

In one embodiment of the invention a threshold of 60 to 80 pg/ml,preferably 70 pg/ml is applied.

In one embodiment of the invention said sample is selected from thegroup comprising human citrate plasma, heparin plasma, EDTA plasma,whole blood.

In one embodiment of the invention said sample taken is directlymeasured without any further sample preparation.

In one embodiment of the invention said method is performed on a fullyautomated device. Roche Elecsys®, Abbott Architect®, Siemens Centauer®,Brahms Kryptor®, Biomerieux Vidas®, Alere Triage®.

In one embodiment of the invention mature ADM 1-52 and/or mature ADM1-52-Gly is determined in at least two samples wherein said samples aretaken in different points of time from said septic patients. Saidsamples may be taken once a day during days of therapy. Such diagnosticregimen may be applied that are described for other biomarker, e.g. (25)and also (26).

In one embodiment of the invention the sample volume measured is less orequal to 50 ul.

The in vitro method for therapy follow-up in septic patients accordingto the present invention may be combined with further clinical and/orlaboratory parameters and or clinical scores such as for instance Apache2 score, SOFA score, or others, or one or more parameters containedwithin the score. Variables/parameters maybe combined continuously ordiscontinuously using standard statistical tools.

Subject matter of the invention is further an assay for determiningmature adrenomedullin and/or adrenomedullin-Gly in a sample comprisingtwo binders that bind to two different regions within the region ofmature adrenomedullin and/or adrenomedullin-Gly that is aminoacid21-52-amid SEQ ID No. 1 or aminoacid 21-52-Gly of mature adrenomedullinSEQ ID No. 2 wherein each of said regions comprises at least 4 or 5amino acids and wherein said assay is not a manualcoated-tube-Akridiniumester sandwich assay.

Subject matter of the invention is further an assay for determiningmature adrenomedullin and/or adrenomedullin-Gly and/oradrenomedullin-Gly in a sample comprising two binders that bind to twodifferent regions within the region of mature adrenomedullin and/oradrenomedullin-Gly that is aminoacid 21-52-amid SEQ ID No. 1 oraminoacid 21-52-Gly of mature adrenomedullin SEQ ID No. 2 wherein eachof said regions comprises at least 4 or 5 amino acids and wherein saidassay is a manual coated-tube-Akridiniumester sandwich assay and whereinone of said binders is an antibody binding to SEQ ID No. 4 and whereinthe second of said binders is an antibody binding to SEQ ID No. 7(APRSKISPQGY-CO—NH2).

In one embodiment of the assays for determining mature adrenomedullinand/or adrenomedullin-Gly in a sample according to the present inventionone of said binders binds to a region comprised within the followingsequence of mature ADM:

-   -   CTVQKLAHQIYQ (SEQ ID No. 6)    -   and wherein said second of these binders binds to a region        comprised within the following sequence of mature ADM:    -   APRSKISPQGY (SEQ ID No. 7)

In one embodiment of the assays for determining mature adrenomedullinand/or adrenomedullin-Gly in a sample according to the present inventionthe assay sensitivity of said assay is able to quantify the ADM ofhealthy subjects and is <10 pg/ml, preferably <40 pg/ml and morepreferably <70 pg/ml.

In one embodiment of the assays for determining mature adrenomedullinand/or adrenomedullin-Gly in a sample according to the present inventionsaid binder exhibits an binding affinity to adrenomedullin of at least10⁷ M⁻¹, preferred 10⁸ M⁻¹, preferred affinity constant is greater than10⁹ M⁻¹, most preferred greater than 10¹⁰ M⁻¹. A person skilled in theart knows that it may be considered to compensate lower affinity byapplying a higher dose of compounds and this measure would not leadout-of-the-scope of the invention. Binding affinity may be determined asdescribed above.

In one embodiment of the assays for determining mature adrenomedullinand/or adrenomedullin-Gly in a sample according to the present inventionsaid binder is selected from the group comprising an anti-adrenomedullinantibody or an anti-ADM antibody fragment binding to ADM or a non-Igscaffold binding to adrenomedullin.

In one embodiment of the assays for determining mature adrenomedullinand/or adrenomedullin-Gly in a sample according to the present inventionsuch assay is a sandwich assay, preferably a fully automated assay. Itmay be an ELISA fully automated or manual. It may be a so-calledPCT-test (point-of-care). Examples of automated or fully automated assaycomprise assays that may be used for one of the following systems: RocheElecsys®, Abbott Architect®, Siemens Centauer®, Brahms Kryptor®,Biomerieux Vidas®, Alere Triage®. Examples of test formats are providedabove.

In one embodiment of the assays for determining mature adrenomedullinand/or adrenomedullin-Gly in a sample according to the present inventionat least one of said two binders is labeled in order to be detected.Examples of labels are provided above.

In one embodiment of the assays for determining mature adrenomedullinand/or adrenomedullin-Gly in a sample according to the present inventionat least one of said two binders is bound to a solid phase. Examples ofsolid phases are provided above.

In one embodiment of the assays for determining mature adrenomedullinand/or adrenomedullin-Gly in a sample according to the present inventionsaid label is selected from the group comprising chemiluminescent label,enzyme label, fluorescence label, radioiodine label.

A further subject of the present invention is a kit comprising an assayaccording to the present invention wherein the components of said assaymay be comprised in one or more container.

A further subject of the present invention is a method of calibrating anassay according to the present invention wherein binder, preferably anantibody, is used that binds to a region of at least 5 amino acidswithin mature adrenomedullin and/or adrenomedullin-Gly amino acids 1-16(SEQ ID No. 8). Said binder may be an antibody or antibody fragment ornon-Ig scaffold that binds to a region of at least 5 amino acids withinmature adrenomedullin and/or adrenomedullin-Gly amino acids 1-16 (SEQ IDNo. 8).

In one embodiment of the method of calibrating an assay according to theinvention said N-terminal antibody or fragment or scaffold recognizesand binds to the N-terminal end (aa1) of mature adrenomedullin and/oradrenomedullin-Gly. This means in another preferred embodiment saidanti-ADM-antibody or an anti-Adrenomendullin antibody fragment or non-Igscaffold binds only to a region within the sequence of mature ADM if theN-terminal end of ADM is free. In said embodiment the anti-ADM-antibodyor anti-Adrenomendullin antibody fragment or non-Ig scaffold would notbind to a region within the sequence of mature ADM if said sequence iscomprised within pro-ADM.

Antibodies suitable for calibrating an assay according to the inventionare such binders that appease the adsorptive properties of ADM. Furthersuch a binder must be compatible to the binders used in the detectionassay, e.g. said binder should not interfere with the binding of thelabelled binder and the solid phase binder in case of an ELISA.

The present methods and assays are suitable for routine applications.Routine applications require in most cases that the sample volume neededshould not exceed 50 ul. Routine application also requires that thepre-analytical treatments are kept to a minimum or are zero (use ofroutine samples like EDTA plasma, Citrate plasma). Preanalyticalrequirements must fit to clinical routine: minimum analyte stability(>90% recovery) shall be at room temperature at least 2 hours.

An antibody according to the present invention is a protein includingone or more polypeptides substantially encoded by immunoglobulin genesthat specifically binds an antigen. The recognized immunoglobulin genesinclude the kappa, lambda, alpha (IgA), gamma (IgG₁, IgG₂, IgG₃, IgG₄),delta (IgD), epsilon (IgE) and mu (IgM) constant region genes, as wellas the myriad immunoglobulin variable region genes. Full-lengthimmunoglobulin light chains are generally about 25 Kd or 214 amino acidsin length. Full-length immunoglobulin heavy chains are generally about50 Kd or 446 amino acid in length. Light chains are encoded by avariable region gene at the NH2-terminus (about 110 amino acids inlength) and a kappa or lambda constant region gene at the COOH-terminus.Heavy chains are similarly encoded by a variable region gene (about 116amino acids in length) and one of the other constant region genes.

The basic structural unit of an antibody is generally a tetramer thatconsists of two identical pairs of immunoglobulin chains, each pairhaving one light and one heavy chain. In each pair, the light and heavychain variable regions bind to an antigen, and the constant regionsmediate effector functions. Immunoglobulins also exist in a variety ofother forms including, for example, Fv, Fab, and (Fab′)₂, as well asbifunctional hybrid antibodies and single chains ((27), (28), (29),(30), (31)). An immunoglobulin light or heavy chain variable regionincludes a framework region interrupted by three hypervariable regions,also called complementarity determining regions (CDR's) see (32). Asnoted above, the CDRs are primarily responsible for binding to anepitope of an antigen. An immune complex is an antibody, such as amonoclonal antibody, chimeric antibody, humanized antibody or humanantibody, or functional antibody fragment, specifically bound to theantigen.

Chimeric antibodies are antibodies whose light and heavy chain geneshave been constructed, typically by genetic engineering, fromimmunoglobulin variable and constant region genes belonging to differentspecies. For example, the variable segments of the genes from a mousemonoclonal antibody can be joined to human constant segments, such askappa and gamma 1 or gamma 3. In one example, a therapeutic chimericantibody is thus a hybrid protein composed of the variable orantigen-binding domain from a mouse antibody and the constant oreffector domain from a human antibody, although other mammalian speciescan be used, or the variable region can be produced by moleculartechniques. Methods of making chimeric antibodies are well known in theart, e.g., see (33). A “humanized” immunoglobulin is an immunoglobulinincluding a human framework region and one or more CDRs from a non-human(such as a mouse, rat, or synthetic) immunoglobulin. The non-humanimmunoglobulin providing the CDRs is termed a “donor” and the humanimmunoglobulin providing the framework is termed an “acceptor.” In oneembodiment, all the CDRs are from the donor immunoglobulin in ahumanized immunoglobulin. Constant regions need not be present, but ifthey are, they must be substantially identical to human immunoglobulinconstant regions, i.e., at least about 85-90%, such as about 95% or moreidentical. Hence, all parts of a humanized immunoglobulin, exceptpossibly the CDRs, are substantially identical to corresponding parts ofnatural human immunoglobulin sequences. A “humanized antibody” is anantibody comprising a humanized light chain and a humanized heavy chainimmunoglobulin. A humanized antibody binds to the same antigen as thedonor antibody that provides the CDRs. The acceptor framework of ahumanized immunoglobulin or antibody may have a limited number ofsubstitutions by amino acids taken from the donor framework. Humanizedor other monoclonal antibodies can have additional conservative aminoacid substitutions which have substantially no effect on antigen bindingor other immunoglobulin functions. Exemplary conservative substitutionsare those such as gly, ala; val, ile, leu; asp, glu; asn, gin; ser, thr;lys, arg; and phe, tyr. Humanized immunoglobulins can be constructed bymeans of genetic engineering (e.g., see (34)). A human antibody is anantibody wherein the light and heavy chain genes are of human origin.Human antibodies can be generated using methods known in the art. Humanantibodies can be produced by immortalizing a human B cell secreting theantibody of interest. Immortalization can be accomplished, for example,by EBV infection or by fusing a human B cell with a myeloma or hybridomacell to produce a trioma cell. Human antibodies can also be produced byphage display methods (see, e.g., (35), (36), (37), which are hereinincorporated by reference), or selected from a human combinatorialmonoclonal antibody library (see the Morphosys website). Humanantibodies can also be prepared by using transgenic animals carrying ahuman immunoglobulin gene (for example, see (38) and (39) which areherein incorporated by reference).

Thus, the ADM antibody may have the formats known in the art. Examplesare human antibodies, monoclonal antibodies, humanized antibodies,chimeric antibodies, CDR-grafted antibodies. In a preferred embodimentantibodies according to the present invention are recombinantly producedantibodies as e.g. IgG, a typical full-length immunoglobulin, orantibody fragments containing at least the F-variable domain of heavyand/or light chain as e.g. chemically coupled antibodies (fragmentantigen binding) including but not limited to Fab-fragments includingFab minibodies, single chain Fab antibody, monovalent Fab antibody withepitope tags, e.g. Fab-V5Sx2; bivalent Fab (mini-antibody) dimerizedwith the CH3 domain; bivalent Fab or multivalent Fab, e.g. formed viamultimerization with the aid of a heterologous domain, e.g. viadimerization of dHLX domains, e.g. Fab-dHLX-FSx2; F(ab′)2-fragments,scFv-fragments, multimerized multivalent or/and multispecificscFv-fragments, bivalent and/or bispecific diabodies, BITE® (bispecificT-cell engager), trifunctional antibodies, polyvalent antibodies, e.g.from a different class than G; single-domain antibodies, e.g. nanobodiesderived from camelid or fish immunoglobulines and numerous others.

In addition to anti-ADM antibodies other biopolymer scaffolds are wellknown in the art to complex a target molecule and have been used for thegeneration of highly target specific biopolymers. Examples are aptamers,spiegelmers, anticalins and conotoxins.

In a preferred embodiment the ADM antibody format is selected from thegroup comprising Fv fragment, scFv fragment, Fab fragment, scFabfragment, (Fab)2 fragment and scFv-Fc Fusion protein. In anotherpreferred embodiment the antibody format is selected from the groupcomprising scFab fragment, Fab fragment, scFv fragment andbioavailability optimized conjugates thereof, such as PEGylatedfragments. One of the most preferred formats is the scFab format.

Non-Ig scaffolds may be protein scaffolds and may be used as antibodymimics as they are capable to bind to ligands or antigenes. Non-Igscaffolds may be selected from the group comprising tetranectin-basednon-Ig scaffolds (e.g. described in (40), fibronectin scaffolds (e.g.described in (41); lipocalin-based scaffolds ((e.g. described in (42));ubiquitin scaffolds (e.g. described in (43)), transferring scaffolds(e.g. described in (44)), protein A scaffolds (e.g. described in (45)),ankyrin repeat based scaffolds (e.g. described in (46), microproteinspreferably microproteins forming a cystine knot) scaffolds (e.g.described in (47)), Fyn SH3 domain based scaffolds (e.g. described in(48) EGFR-A-domain based scaffolds (e.g. described in (49)) and Kunitzdomain based scaffolds (e.g. described in (59)).

In another preferred embodiment the anti-ADM antibody or the antibodyfragment binding to ADM is a monospecific antibody. Monospecificantibodies are antibodies that all have affinity for the same antigen.Monoclonal antibodies are monospecific, but monospecific antibodies mayalso be produced by other means than producing them from a common germcell.

In one preferred embodiment of the invention antibodies according to thepresent invention may be produced as follows:

A Balb/c mouse was immunized with 100 μg ADM-Peptide-BSA-Conjugate atday 0 and 14 (emulsified in 100 μl complete Freund's adjuvant) and 50 μgat day 21 and 28 (in 100 μl incomplete Freund's adjuvant). Three daysbefore the fusion experiment was performed, the animal received 50 μg ofthe conjugate dissolved in 100 μl saline, given as one intraperitonealand one intra venous injection.

Splenocytes from the immunized mouse and cells of the myeloma cell lineSP2/0 were fused with 1 ml 50% polyethylene glycol for 30 s at 37° C.After washing, the cells were seeded in 96-well cell culture plates.Hybrid clones were selected by growing in HAT medium [RPMI 1640 culturemedium supplemented with 20% fetal calf serum and HAT-Supplement]. Aftertwo weeks the HAT medium is replaced with HT Medium for three passagesfollowed by returning to the normal cell culture medium.

The cell culture supernatants were primary screened for antigen specificIgG antibodies three weeks after fusion. The positive testedmicrocultures were transferred into 24-well plates for propagation.After retesting the selected cultures were cloned and recloned using thelimiting-dilution technique and the isotypes were determined (see also(51) and (52)).

Antibodies may be produced by means of phage display according to thefollowing procedure:

The human naive antibody gene libraries HAL7/8 were used for theisolation of recombinant single chain F-Variable domains (scFv) againstadrenomedullin peptide. The antibody gene libraries were screened with apanning strategy comprising the use of peptides containing a biotin taglinked via two different spacers to the adrenomedullin peptide sequence.A mix of panning rounds using non-specifically bound antigen andstreptavidin bound antigen were used to minimize background ofnon-specific binders. The eluted phages from the third round of panninghave been used for the generation of monoclonal scFv expressing E. colistrains. Supernatant from the cultivation of these clonal strains hasbeen directly used for an antigen ELISA testing. (53) and (54).

Humanization of murine antibodies may be conducted according to thefollowing procedure:

For humanization of an antibody of murine origin the antibody sequenceis analyzed for the structural interaction of framework regions (FR)with the complementary determining regions (CDR) and the antigen. Basedon structural modeling an appropriate FR of human origin is selected andthe murine CDR sequences are transplanted into the human FR. Variationsin the amino acid sequence of the CDRs or FRs may be introduced toregain structural interactions, which were abolished by the speciesswitch for the FR sequences. This recovery of structural interactionsmay be achieved by random approach using phage display libraries or viadirected approach guided by molecular modeling (55.)

Development of Antibodies

We developed mouse monoclonal antibodies binding to the N-terminal,mid-regional and C-terminal part of hADM and their affinity constantswere determined (table 1).

Peptides for Immunization

Peptides were supplied by JPT Peptide Technologies GmbH (Berlin,Germany). Peptides were coupled to BSA using the Sulfo-SMCC crosslinkingmethod. The crosslinking procedure was performed according themanufacturers instructions (Thermo Fisher/Pierce).

EXAMPLE 1 Generation of Antibodies and Determination of their AffinityConstants

The murine antibodies were generated according to the following method:

A Balb/c mouse was immunized with 100 μg Peptide-BSA-Conjugate at day 0and 14 (emulsified in 100 μl complete Freund's adjuvant) and 50 μg atday 21 and 28 (in 100 μl incomplete Freund's adjuvant). Three daysbefore the fusion experiment was performed, the animal received 50 μg ofthe conjugate dissolved in 100 μl saline, given as one intraperitonealand one intra venous injection.

Splenocytes from the immunized mouse and cells of the myeloma cell lineSP2/0 were fused with 1 ml 50% polyethylene glycol for 30 s at 37° C.After washing, the cells were seeded in 96-well cell culture plates.Hybrid clones were selected by growing in HAT medium [RPMI 1640 culturemedium supplemented with 20% fetal calf serum and HAT-Supplement]. Aftertwo weeks the HAT medium is replaced with HT Medium for three passagesfollowed by returning to the normal cell culture medium.

The cell culture supernatants were primary screened for antigen specificIgG antibodies three weeks after fusion. The positive testedmicrocultures were transferred into 24-well plates for propagation.After retesting the selected cultures were cloned and recloned using thelimiting-dilution technique and the isotypes were determined. ((51) and(52)).

TABLE 1 ADM Affinity constants Antigen/Immunogen Region DesignationKd (M⁻¹) YRQSMNNFQGLRSFGC 1-16 NT-ADM 1.6 × 10⁹ CTVQKLAHQIYQ 21-32MR-ADM   2 × 10⁹ CAPRSKISPQGY-NH₂ C-42-52 CT-ADM 1.1 × 10⁹

Monoclonal Antibody Production

Antibodies were produced via standard antibody production methods (56)and purified via Protein A. The antibody purities were >95% based on SDSgel electrophoresis analysis.

Affinty Constants

To determine the affinity of the antibodies to Adrenomedullin, thekinetics of binding of Adrenomedullin to immobilized antibody wasdetermined by means of label-free surface plasmon resonance using aBiacore 2000 system (GE Healthcare Europe GmbH, Freiburg, Germany).Reversible immobilization of the antibodies was performed using ananti-mouse Fc antibody covalently coupled in high density to a CM5sensor surface according to the manufacturer's instructions (mouseantibody capture kit; GE Healthcare). (22)

Labelling procedure (tracer): 100 ug (100 ul) of antibody (1 mg/ml inPBS, pH 7.4,) was mixed with 10 ul Akridinium NHS-ester (1 mg/ml inacetonitrile, InVent GmbH, Germany) (57) and incubated for 20 min atroom temperature. Labelled CT-H was purified by Gel-filtration HPLC onBio-Sil® SEC 400-5 (Bio-Rad Laboratories, Inc., USA) The purifiedlabeled antibody was diluted in (300 mmol/L potassiumphosphate, 100mmol/L NaCl, 10 mmol/L Na-EDTA, 5 g/L Bovine Serum Albumin, pH 7.0). Thefinal concentration was approx. 800.000 relative light units (RLU) oflabelled compound (approx. 20 ng labeled antibody) per 200 μL.Akridiniumester chemiluminescence was measured by using an AutoLumat LB953 (Berthold Technologies GmbH & Co. KG).

Solid phase: Polystyrene tubes (Greiner Bio-One International AG,Austria) were coated (18 h at room temperature) with antibody ((1.5 μgantibody/0.3 mL 100 mmol/L NaCl, 50 mmol/L TRIS/HCl, pH 7.8). Afterblocking with 5% bovine serum albumine, the tubes were washed with PBS,pH 7.4 and vacuum dried.

-   -   Calibrators:    -   Synthetic human ADM (Bachem, Switzerland) was linearily diluted        using 50 mM Tris/HCl, 250 mM NaCl, 0.2% Triton X-100, 0.5% BSA,        20 tabs/L Protease cOmplete Protease Inhibitor Cocktail Tablets        (Roche AG); pH 7.8. Calibrators were stored at −20° C. before        use.

EXAMPLE 2 Determination of the Antibody Combination that Yields HighSignal/Noise Ratios

hADM Immunoassay:

50 ul of sample (or calibrator) was pipetted into coated tubes, afteradding labeled second antibody (200 ul), the tubes were incubated for 2h at room temperature. Unbound tracer was removed by washing 5 times(each 1 ml) with washing solution (20 mM PBS, pH 7.4, 0.1% TritonX-100).

Tube-bound chemiluminescence was measured by using the LB 953

All antibodies were used in a sandwich immunoassay as coated tube andlabeled antibody and combined in the following variations (table 2):

Incubation was performed as described under hADM-Immunoassay. Resultsare given in ratio of specific signal (at 10 ng/ml ADM)/background(sample without ADM) signal.

TABLE 2 Signal/noise ratio NT-ADM tracer MR-ADM tracer CT-ADM tracerNT-ADM / 195 241 MRADM 204 / 904 CT-ADM 260 871 /

Surprisingly, we found the combination of MR-ADM and CT-ADM ascombination for highest signal/noise ratio.

Subsequently, we used this antibody-combination for furtherinvestigations. We used MR-ADM as solid phase antibody and CT-ADM aslabeled antibody. A typical dose/signal curve is shown in FIG. 1. Theanalytical sensitivity (average of 10 runs, ADM-free sample+2SD) of theassay was 2 μg ADM/ml.

EXAMPLE 3 Stability of Human Adrenomedullin

Human ADM was diluted in human Citrate plasma (n=5, final concentration10 ng ADM/ml) and incubated at 24° C. At selected time points, aliquotswere frozen at −20° C. Immediately after thawing the samples hADM wasquantified by using the hADM immunoassay described above.

Table 3 shows the stability of hADM in human plasma at 24° C.

Average ADM Relative loss of Loss of immune Time (h) recovery (N = 5)immune reactivity reactivity %/h 0 100 / / 2 99.2 0.8 0.4 4 96.4 3.6 0.88 88.2 11.8 1.5 Average: 0.9%/h

Surprisingly, using the antibody-combinations MR-ADM and CT-ADM in asandwich immune assay, the preanalytical stability of the analyte ishigh (only 0.9%/h average loss of immune reactivity). In contrast, usingother assay methods, a plasma half life of only 22 min was reported(Hinson 2000). Since the time from taking sample to analysis in hospitalroutine is less than 2 h, the used ADM detection method is suitable forroutine diagnosis. It is remarkable, that any non routine additives tosamples (like Aprotinin, (20)) are not needed to reach acceptableADM-immune reactivity stabilities.

EXAMPLE 4 Reproducibility of Calibrator-Preparations

We found a high variation of results, preparing calibrators for ADMassays (average CV 8.5%, see table 4). This may be due to highadsorption of hADM to plastic and glass surfaces (see also (58). Thiseffect was only slightly reduced by adding detergents (up to 1% Triton X100 or 1% Tween 20), protein (up to 5% BSA) and high ionic strength (upto 1M NaCl) or combinations thereof. Surprisingly, if a surplus of antiADM antibody (10 ug/ml) is added to the calibrator dilution buffer, therecovery and reproducibility of ADM assay calibrator-preparations wassubstantially improved to <1% of inter preparation CV (table 4).

Fortunately, the presence of N-terminal antibodies did not effect theADM-signal generated by the combination of MR- and C-terminal antibodies(FIG. 11).

TABLE 4 In the presence of Inter Inter NT-ADM antibody preparationWithout preparation calibrator (10 ug/ml) CV (%) antibody CV (%) 100ng/ml 3453 s/n-r 0.9 2842 s/n-r 2.8  10 ng/ml 1946 s/n-r 0.8 1050 s/n-r7.9  1 ng/ml  179 s/n-r 1.1  77 s/n-r 14.8 Average: 0.93 Average: 8.5

Inter Preparation Variation of Calibrators.

ADM assay calibrators were prepared as described above with and without10 ug/ml of NT-ADM-antibody. Coefficients of variation are given from 5independent preparation runs. The calibrators were measured using theADM assay described above. s/n-r=signal to noise ratio.

For all following studies, we used an ADM assay, based on calibrators,prepared in the presence of 10 ug/ml of NT-ADM antibody and 10 ug/ml ofNT-ADM antibody as supplement in the tracer buffer.

EXAMPLE 5 Sensitivity

The goal of assay sensitivity was to completely cover the ADMconcentration of healthy subjects.

ADM Concentration in Healthy Subjects:

Healthy subjects (n=100, average age 56 years) were measured using theADM assay. The median value was 24.7 pg/ml, the lowest value 11 pg/mland the 99^(th) percentile 43 pg/ml. Since the assay sensitivity was 2pg/ml, 100% of all healthy subjects were detectable using the describedADM assay (see FIG. 2).

EXAMPLE 6 Clinical Study

101 ED patients fulfilling the definition of sepsis (59) weresubsequently hospitalized (average 5 days of hospitalization) andreceived a standard of care treatment. EDTA-plasma was generated fromday 1 (ED presentation) and one sample each day during hospital stay.The time to freeze samples for later ADM-measurement was less than 4 h.

Patient characteristics are summarized in table 5

TABLE 5 all in hospital deaths discharged Variable (n = 101) (n = 27) (n= 74) p-value Demographics Gender - male 60 (60) 13 (48) 47 (64) 0.163Age - median [IQR] 78 [72-72] 77 [71.25-83] 80 [75-84.5] 0.142Examination variables BP systolic (mmHg) - median [IQR] 115 [100-100]120 [106.25-138.75] 105 [80-120] 0.001 BP diastolic (mmHg) - median[IQR] 65 [60-60] 65 [60-85] 60 [50-70] 0.002 HR - median [IQR] 100[94-94] 100 [94-114.75] 100 [93.5-107.5] 0.407 RR - median [IQR] 24[22-22] 24 [22-28] 26 [24-28] 0.069 MAP (mmHg) - median [IQR] 83.3[74-74] 83.3 [77.62-100.75] 81.6 [63.5-89] 0.026 concomitant diseasesCardiovascular - yes 26 (25.7) 9 (33.3) 17 (23) 0.311 Hypertensive - yes47 (46.5) 13 (48.1) 34 (45.9) 1.000 Diabetes - yes 35 (34.7) 9 (33.3) 26(35.1) 1.000 Cancere - yes 13 (12.9) 3 (11.1) 10 (13.5) 1.000 routinelabaratory variables Blood culture - yes 31 (31) 5 (19) 26 (35) 0.246negative 15 (16.3) 2 (8) 13 (19.4) positive 16 (17.4) 3 (12) 13 (19.4)Creatinine clearance (ml/min) - 48 [23.25-23.25] 56 [29.25-80] 31.5[14.75-66] 0.043 median [IQR] Creatinine - median [IQR] 1.3 [0.9-0.9]1.25 [0.9-2.08] 1.8 [1-3.15] 0.080 UREA - median [IQR] 36 [21-21] 31.5[20-53.25] 51 [42-87] 0.004 GCS - median [IQR] 15 [10-10] 15 [12.5-15] 8[8-11] <0.001 Pcr - median [IQR] 16 [6.6-6.6] 14.5 [6.7-23.7] 17.35[6.6-28.05] 0.846 Gluco - median [IQR] 113.5 [94.5-94.5] 110 [95.5-144]128 [94-160.5] 0.400 biliru - median [IQR] 0.9 [0.71-0.71] 0.9[0.7-1.03] 0.91 [0.77-1.18] 0.534 GR - median [IQR] 3.8 [3.3-3.3] 3.8[3.2-4.3] 3.7 [3.4-4.2] 0.684 GB - median [IQR] 12700 [6774-6774] 13100[8115-17565] 11920 [25.55-18790] 0.343 PLT - median [IQR] 213 [150-150]217 [154.75-301] 185 [130-236.5] 0.113 HCT - median [IQR] 32 [28-28]31.5 [28-37] 34 [31.25-39.5] 0.149 Leuco/Neutr (%) - median [IQR] 87[80-80] 86 [78.25-89.95] 91 [87-93.05] 0.001 HB - median [IQR] 10.4[9.47-9.47] 10.15 [9.3-12.4] 10.85 [9.9-12.67] 0.220 Na - median [IQR]137 [134-134] 137 [133-141] 139 [134-144.5] 0.204 K - median [IQR] 3.9[3.5-3.5] 3.9 [3.6-4.3] 3.9 [3.3-5.1] 0.982 INR - median [IQR] 1.19[1.1-1.1] 1.19 [1.1-1.4] 1.18 [1.04-1.36] 0.731 TC - median [IQR] 38.4[36-36] 38.5 [38.12-38.7] 36 [35.55-38.5] <0.001 SAO2 - median [IQR] 94[90-90] 95 [90.25-97] 93 [88.5-95.5] 0.119 pH - median [IQR] 7.45[7.38-7.38] 7.46 [7.4-7.5] 7.4 [7.24-7.4] <0.001 PO2 - median [IQR] 67[56-56] 66.5 [56-78] 67 [56.5-79.5] 0.806 PCO2 - median [IQR] 36 [32-32]37.5 [33-43.75] 34 [30-41] 0.245 Lact - median [IQR] 1.5 [1-1] 1.3[0.83-1.9] 2.5 [1.4-4.15] <0.001 Bic - median [IQR] 23.5 [21-21] 24.25[21.43-28] 21 [17.35-23.25] 0.001 FiO2 (%) - median [IQR] 21 [21-21] 21[21-23.25] 24 [21-45] <0.001 other Acute organ disfunction - yes 39(43.3) 16 (64) 23 (35.4) 0.021 Apache score (%) - median [IQR] 19[12.5-12.5] 14.65 [12.12-20.38] 32 [20-39] <0.001 Days hospitalized -median [IQR] 5 [2-2] 6 [4-7] 2 [1-6] 0.003 treatment at baselineDiuresis (cc) - median [IQR] 900 [600-600] 1000 [700-1200] 450[200-1025] <0.001 Steroids - yes 16 (15.8) 4 (14.8) 12 (16.2) 1.000Vasopressors - yes 18 (17.8) 13 (48.1) 5 (6.8) <0.001 Antibiotics - yes101 (100) 27 (100) 74 (100) 1.000 Fluid therapy - yes 101 (100) 27 (100)74 (100) 1.000 new biomarker ADM (pg/mL) - median [IQR] 53.8 [37.4-94.0]93.9 [48.7-241] 50.1 [32.2-77.5] <0.001

26.7% of all patients died during hospital stay and are counted astreatment non responder, 73.3% of all patients survived the sepsis andare counted as treatment responder.

66% off all patients presenting with sepsis had a non-normal ADMvalue >43 pg/ml (99^(th) percentile), indicating ADM not to be a markerfor the infection.

Results of Clinical Study Initial ADM is Highly Prognostic.

We correlated the initial ADM value with the in hospital mortality andcompared ADM with APACHE 2 sepsis score (see (60)). ADM is highlyprognostic for sepsis outcome (see FIG. 3) and comparable to APACHE 2score. There is a significant added information if ADM and APACHE 2 arecombined (FIG. 4).

ADM in Treatment Monitoring.

Patients were treated based on standard of care treatments (table 5).The average hospitalization time was 5 days. ADM was measured each dayin hospital (day 1=admission) and correlated to in hospital mortality(table 6). ADM changed during hospital stay and the change during timeimproved the prognostic value by 52% from initial Chi² of 19.2 to 29.2on day 5.

Using a simple cut off model at 70 pg/ml of ADM showed a 68% risk ofdeath for patients starting at ADM concentrations >70 pg/ml and remainall the hospital stay >70 pg/ml (treatment non-responder). Patientshaving all time an ADM value <70 pg/ml or developing from >70 pg/ml to<70 pg/ml had a mortality of only 11% (well treated/treatment responder)and patients presenting with ADM values >70 pg/ml and reducing their ADMconcentration during hospital treatment to values <70 pg/ml had a 0%mortality. There were no patients developing from <70 pg/ml to >70 pg/mlduring hospital treatment. The average time needed to generateresponder/nonresponder information for all patients was about 1 day.The >70 pg/ml—patients responding to treatment during hospital stayneeded about 2 days to indicate treatment success by ADM.

TABLE 6 Patients Patient all Patient changed from days >70 all days >70pg/ml to pg/ml <70 pg/ml <70 pg/ml n 28/101 73/101 15/73 (27.7%) (72.3%)(20.5%) Mortality 68% 11% 0% Average days after 1 day 1.2 days 2.2 dayshospitalization of change from ADM >70 pg/ml to ADM <70 pg/ml or nochange

LITERATURE

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FIGURE DESCRIPTION

FIG. 1 shows a typical hADM dose/response curve using MR-ADM as solidphase antibody and CT-ADM as labeled antibody.

FIG. 2: Healthy subjects (n=100, average age 56 years) were measuredusing the ADM assay. The median value was 24.7 pg/ml, the lowest value11 pg/ml and the 99^(th) percentile 43 pg/ml. Since the assaysensitivity was 2 pg/ml, 100% of all healthy subjects were detectableusing the described ADM assay

FIG. 3: Predicting in-hospital mortality—Results from logisticregression

FIG. 4: Predicting in-hospital mortality—ADM is independent from Apacheand provides additional prognostic information

FIGS. 5 to 10: Individual patient ADM-kinetics

FIG. 5 and FIG. 6: Survivors, ADM is <70 pg/ml at ED presentation(day 1) and during hospital stay. ADM indicates a well treated patient.

FIG. 7 and FIG. 8: Survivors, ADM is above 70 pg/ml at ED presentation(day 1) and is lowered during hospital treatment to values below 70pg/ml (treatment responder).

FIG. 9 and FIG. 10: Deceased patients, ADM is above 70 pg/ml at EDpresentation (day 1) and is not lowered to values below 70 pg/ml duringhospital treatment (treatment non responder).

FIG. 11: ADM assay in the presence and absence of N-terminal antibodies

The ADM assay was performed as described above. A) reference curve B) inthe presence of NT-ADM-antibody (10 ug/ml, 3.33 ug/test). The additionof NT-ADM-antibody did not influence the ADM assay.

SEQUENCES SEQ ID No. 1: ADM 21-52 CTVQKLAHQIYQFTDKDKDNVAPRSKISPQGY-CONH2SEQ ID No. 2: ADM 21-52-Gly CTVQKLAHQIYQFTDKDKDNVAPRSKISPQGYGSEQ ID No. 3: PreProADM MKLVSVALMYLGSLAFLGADTARLDVASEFRKKWNKWALSRGKRELRMSSSYPTGLADVKAGPAQTLIRPQDMKGASRSPEDSSPDAARIRVKRYRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVAPRSKISPQGYGRRRRRSLPEAGPGRTLVSSKPQAHGAPAP LPSGSAPHFSEQ ID No. 4: ADM 1-52 YRQSMNNFQGLKSFGCRFGTCTVQKLAHQIYQFTDKDKDNVAPRSKISPQGY-CONH2 SEQ ID No. 5: ADM 1-52-GlyYRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVAPR SKISPQGYGSEQ ID No. 6: ADM 21-32 CTVQKLAHQIYQ SEQ ID No. 7: ADM 42-52 APRSKISPQGYSEQ ID No. 8: ADM 146-Gly (amino acid) YRQSMNNFQGLRSFGC

1. An in vitro method for therapy follow-up in patients being suspectedof having sepsis wherein the concentration of mature ADM 1-52 and/ormature ADM 1-52-Gly in a sample of bodily fluid of said septic patientis determined using an assay comprising two binders that bind to twodifferent regions within the region of mature adrenomedullin and/oradrenomedullin-Gly that is aminoacid 21-52-amid (SEQ ID No. 1) oraminoacid 21-52-Gly (SEQ ID No. 2) wherein each of said regionscomprises at least 4 or 5 amino acids.
 2. An in vitro method for therapyfollow-up in septic patients according to claim 1 wherein one of saidbinders binds to a region comprised within the following sequence ofmature ADM and/or mature ADM 1-52-Gly (SEQ ID No. 4) and wherein saidsecond of these binders binds to a region comprised within the followingsequence of mature ADM and/or mature ADM 1-52-Gly (SEQ ID No. 5).
 3. Anin vitro method for therapy follow-up in septic patients according toclaim 1 wherein the assay sensitivity of said assay is able to quantifythe ADM of healthy subjects and is <10 pg/ml, preferably <40 pg/ml andmore preferably <70 pg/ml.
 4. An in vitro method for therapy follow-upin septic patients according to claim 1 wherein said binder exhibits anbinding affinity to mature ADM and/or mature ADM 1-52-Gly of at least10⁷ M⁻¹.
 5. An in vitro method for therapy follow-up in septic patientsaccording to claim 1 wherein said binder is selected from the groupcomprising an anti-adrenomedullin antibody or an anti-ADM antibodyfragment binding to ADM or a non-Ig scaffold binding to adrenomedullin.6. An in vitro method for therapy follow-up in septic patients accordingto claim 1 wherein such assay is a sandwich assay, preferably a fullyautomated assay.
 7. An in vitro method for therapy follow-up in septicpatients according to claim 1 wherein at least one of said two bindersis labeled in order to be detected.
 8. An in vitro method for therapyfollow-up in septic patients according to claim 1 wherein at least oneof said two binders is bound to a solid phase.
 9. An in vitro method fortherapy follow-up in septic patients according to claim 7 wherein saidlabel is selected from the group comprising chemiluminescent label,enzyme label, fluorescence label, radioiodine label.
 10. An in vitromethod for therapy follow-up in septic patients according to claim 1wherein the concentration of mature ADM 1-52 and/or mature ADM 1-52-Glymeasured in the sample is in the range between 10-500 pg/ml.
 11. An invitro method for therapy follow-up in septic patients according to claim1 wherein a threshold is applied whereby a value above threshold isindicative of a patient that is not or bad responding to therapy andwhereas a value below said threshold is indicative of a patientresponding to therapy.
 12. An in vitro method for therapy follow-up inseptic patients according to claim 1 wherein a threshold of 60 to 80pg/ml, preferably 70 pg/ml is applied.
 13. An in vitro method fortherapy follow-up in septic patients according to claim 1 wherein saidsample is selected from the group comprising human citrate plasma,heparin plasma, EDTA plasma, whole blood.
 14. An in vitro method fortherapy follow-up in septic patients according to claim 13 wherein saidsample taken is directly measured without any further samplepreparation.
 15. An in vitro method for therapy follow-up in septicpatients according to claim 1 wherein said method is performed on afully automated device.
 16. An in vitro method for therapy follow-up inseptic patients according to claim 1 wherein mature ADM 1-52 and/ormature ADM 1-52-Gly is determined in at least two samples wherein saidsamples are taken in different points of time from said septic patients.17. An in vitro method for therapy follow-up in septic patientsaccording to claim 1 wherein the sample volume measured is less or equalto 50 ul.
 18. Assay for determining mature adrenomedullin and/oradrenomedullin-Gly and/or adrenomedullin-Gly in a sample comprising twobinders that bind to two different regions within the region of matureadrenomedullin and/or adrenomedullin-Gly that is aminoacid 21-52-amidSEQ ID No. 1 or aminoacid 21-52-Gly of mature adrenomedullin SEQ ID No.2 wherein each of said regions comprises at least 4 or 5 amino acids andwherein said assay is not a manual coated-tube-Akridiniumester sandwichassay.
 19. Assay for determining mature adrenomedullin and/oradrenomedullin-Gly and/or adrenomedullin-Gly in a sample comprising twobinders that bind to two different regions within the region of matureadrenomedullin and/or adrenomedullin-Gly that is aminoacid 21-52-amid(SEQ ID No. 1) or aminoacid 21-52-Gly of mature adrenomedullin (SEQ IDNo. 2) wherein each of said regions comprises at least 4 or 5 andwherein said assay is a manual coated-tube-Akridiniumester sandwichassay and wherein one of said binders is an antibody binding to SEQ IDNo. 6 CTVQKLAHQIYQ and wherein the second of said binders is an antibodybinding to SEQ ID No. 7 APRSKISPQGY-CO—NH2.
 20. Assay for determiningmature adrenomedullin and/or adrenomedullin-Gly in a sample according toclaim 18 wherein one of said binders binds to a region comprised withinthe following sequence of mature ADM 1-52-Gly (SEQ ID No. 4) and whereinsaid second of these binders binds to a region comprised within thefollowing sequence of mature ADM and/or mature ADM 1-52-Gly (SEQ ID No.5).
 21. Assay for determining mature adrenomedullin and/oradrenomedullin-Gly in a sample according to claim 18 wherein the assaysensitivity of said assay is able to quantify the ADM of healthysubjects and is <10 pg/ml, preferably <40 pg/ml and more preferably <70pg/ml.
 22. Assay for determining mature adrenomedullin and/oradrenomedullin-Gly in a sample according to claim 18 wherein said binderexhibits a binding affinity to adrenomedullin of at least 10⁷ M⁻¹. 23.Assay for determining mature adrenomedullin and/or adrenomedullin-Gly ina sample according to claim 18 wherein said binder is selected from thegroup comprising an anti-adrenomedullin antibody or an anti-ADM antibodyfragment binding to ADM or a non-Ig scaffold binding to adrenomedullin.24. Assay for determining mature adrenomedullin and/oradrenomedullin-Gly in a sample according to claim 18 wherein such assayis a sandwich assay, preferably a fully automated assay.
 25. Assay fordetermining mature adrenomedullin and/or adrenomedullin-Gly in a sampleaccording to claim 18 wherein at least one of said two binders islabeled in order to be detected.
 26. Assay for determining matureadrenomedullin and/or adrenomedullin-Gly in a sample according to claim18 wherein at least one of said two binders is bound to a solid phase.27. Assay for determining mature adrenomedullin and/oradrenomedullin-Gly in a sample according to claim 25 wherein said labelis selected from the group comprising chemiluminescent label, enzymelabel, fluorescence label, radioiodine label.
 28. Kit comprising anassay according to claim 18 wherein the components of said assay may becomprised in one or more container.
 29. A method of calibrating an assayaccording to claim 18 wherein a binder is used that binds to a region ofat least 5 amino acids within mature adrenomedullin and/oradrenomedullin-Gly amino acids 1-16 (SEQ ID No. 8).
 30. A method ofcalibrating an assay according to claim 29 wherein binder recognizes andbinds to the N-terminal end of mature adrenomedullin and/oradrenomedullin-Gly.